Antibacterial and antiviral agents and methods of screening for the same

ABSTRACT

A method of screening for compounds useful for inhibiting retrovirus propagation in a host for the retrovirus, wherein the retrovirus primes reverse transcription in the host by binding of a specific host tRNA to retrovirus RNA at at least a pair of separate binding sites on the host tRNA, comprises contacting the specific host tRNA to the retrovirus RNA in the presence of the test compound, and then determining whether the compound inhibits the binding of the specific host tRNA to the retrovirus RNA in the presence of the test compound.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/315,674, filed May 20, 1999, now issued as U.S. Pat. No. 6,461,815,which in turn claims the benefit of U.S. provisional application Ser.No. 60/086,380, filed May 22, 1998, the disclosures of which areincorporated by reference herein in their entirety.

This invention was made with government support under grant numberGM23037 from the National Institutes of Health and grant number MCB9631103 from the National Science Foundation. The Government has certainrights to this invention.

FIELD OF THE INVENTION

The present invention concerns antibacterial and antiviral agents thatare directed against tRNA targets, and methods of screening forantibacterial and antiviral agents directed against tRNA targets.

BACKGROUND OF THE INVENTION

Numerous common bacteria are capable of mutating to become resistant toantibiotic agents. Viruses such as HIV are also known to mutate rapidlyand thereby avoid immune defense mechanisms. Accordingly, there is acontinued need for new antimicrobial agents and antiviral agents.

J. Hill et al., PCT Application WO 9705132, describe compounds thatinhibit isoleucyl-tRNA synthetases. The compounds are stated to beuseful against a broad spectrum of bacteria, fungi and parasites.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of inhibitingmicrobe propagation. The method comprises inhibiting ribosomal bindingof a specific microbial tRNA such as tRNA^(lys) _(SUU), tRNA^(gln)_(SUG) or tRNA^(glu) _(SUC) (for example, at position 27-43, mostpreferably position 34, thereof ) in the microbe by an amount sufficientto inhibit microbe propagation. This is achieved by a drug or compoundbinding to positions 27-43 (one or more positions simultaneously), mostpreferably position 34 or 37 of the tRNA. Additionally, the compound ordrug can inactive (in the microbe) one or more enzymes that areresponsible for producing the modification in the specific microbialtRNA that is responsible for the specific binding properties thereof (atpositions 27 to 43, preferably positions 34 or 43).

A second aspect of the present invention is a method of inhibitingretrovirus propagation in a host for that retrovirus, wherein thatretrovirus primes reverse transcription by binding of a specific hosttRNA to retrovirus RNA at at least a pair of separate binding sites. Themethod comprises inhibiting the binding of the specific host tRNA to theretrovirus RNA at one of the binding sites by an amount sufficient toinhibit propagation of the retrovirus in the host. Advantageously,mutation of the retrovirus RNA to bind an alternate host tRNA forpriming of reverse transcription requires a pair of mutations at atleast the pair of separate tRNA binding sites in the retrovirus RNA: alow probability event.

A third aspect of the present invention is a method of screening forcompounds useful for inhibiting microbial propagation. The methodcomprises contacting a specific microbial tRNA (for example, tRNA^(lys)_(SUU)) to a ribosome that binds that tRNA in the presence of the testcompound, and then determining whether the compound inhibits the bindingof that tRNA (e.g., binding of tRNA^(lys) _(SUU), tRNA^(gln) _(SUG) ortRNA^(glu) _(SUC) at position 27-43, most preferably position 34,thereof) to the ribosome. The inhibition of binding indicates that thetest compound is useful for inhibiting microbial propagation. The testcompound will generally be binding to position 27-43, individually or incombination, most preferably positions 34 and or 37 of the stated tRNAs.The screening step may be carried out in a host cell, wherein thecorresponding host cell tRNA (i.e., the tRNA that binds to the sameamino acid) is modified differently as compared to said microbial tRNAat position 27 through 43 thereof (i.e., has a different modification,is not modified where the microbial tRNA is modified, or has the samemodification but in a structure not recognized or specifically bound bythe test compound).

A fourth aspect of the invention is a method of screening for compoundsuseful for inhibiting retrovirus propagation in a host for theretrovirus, wherein the retrovirus primes reverse transcription in thehost by binding of a specific host tRNA to retrovirus RNA at at least apair of separate binding sites on the host tRNA. The method comprisescontacting the specific host tRNA to the retrovirus RNA in the presenceof the test compound, and then determining whether the compound inhibitsthe binding of the specific host tRNA to the retrovirus RNA in thepresence of the test compound. The inhibition of binding indicates thatthe test compound is useful for inhibiting propagation of the virus inthe host. Again, note that mutation of the retrovirus RNA to bind analternate host tRNA for priming of reverse transcription requires atleast a pair of separate mutations at different tRNA binding sites inthe retrovirus RNA.

The foregoing and other objects and aspects of the present invention areexplained in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide sequences of human tRNA^(Lys3) anticodon stem/loopsand structures of modified uridines. A: Nucleotide sequences of theanticodon stem/loops (ASLs) of native human tRNA^(Lys3) _(SUU) and thatof the chemically synthesized tRNA^(Lys) ASLs used in the experiments:ASL^(Lys3) _(SUU) and ASL^(Lys3) _(SUU). B: Chemical structures ofmcm⁵s²U (modified U₃₄ in human tRNA^(Lys3) _(SUU)), mnm⁵s²U (modifiedU₃₄ in E. coli tRNA^(Lys) _(SUU)), and s²U in ASL^(Lys3) _(SUU).Modified nucleosides: ψ=pseudouridine;mcm⁵s²U=5-methoxycarbonylmethyl-2-thiouridine;mnm⁵s²U=5-methylaminomethyl-2-thiouridine;ms²t⁶A=2-methylthio-N⁶-threonylcarbamoyl-adenosine; s²U=2-thiouridine.Human and E. coli tRNA^(Lys) _(SUU) anticodon stem-loop domains haveidentical sequences except for the three base pairs at the top of thestem (P. Agris et al., RNA 3: 420-428 (1997).

FIG. 2. Binding of tRNAs and anticodon/stem loops (ASLs) to programmed30S ribosomal subunits. Ribosome binding assays were carried out asdescribed under Materials and Methods by using ³²P-labeled tRNAs or ASLsand appropriately programmed 30S ribosomal subunits. A: Binding oflysine tRNAs and ASLs to poly(A)-programmed 30S ribosomal subunits:(filled square) native E. coli tRNA^(Lys) _(SUU); (open square) humantRNA^(Lys3) _(UUU) transcript; (filled circle) ASL^(Lys3) _(SUU); and(open circle) ASL^(Lys3) _(UUU). B: Binding of (filled squre) E. colitRNA^(Glu) _(SUC), and (open diamond) E. coli tRNA^(Glu) _(UUC)transcript to poly(AG)-programmed 30S ribosomal subunits, and (opentriangle) E. coli tRNA^(Gln) _(UUG) transcript to poly(AC)-programmed30S ribosomal subunits. C: Binding of (filled square), native yeasttRNA^(Phe) _(GmAA) and (opened square), ASL^(Phe) _(GAA) topoly(U)-programmed 30S ribosomal subunits. G_(m)=2′O -methylguanosine.

FIG. 3. Footprinting of tRNAs and ASLs at the 16S rRNA P-site. A:Chemical probing experiments were conducted with kethoxal, as describedunder Materials and Methods, with: poly(U)- (lanes 1-3) orpoly(A)-programmed (lanes 4-7) 30S ribosomal subunits plus native yeasttRNA^(Phe) _(GmAA) (lane 2); ASL^(Phe) _(GAA) (lane 3); E. colitRNA^(Lys) ^(SUU) (lane 5); ASL^(Lys3) _(SUU) (lane 6); or ASL^(Lys3)_(UUU) (lane 7). B: Dimethyl sulfate protection experiments wereconducted with poly(A)-programmed (lanes 1-4) 30S ribosomal subunitsplus E. coli tRNA^(Lys) _(SUU) (lane 2); ASL^(Lys3) _(SUU) (lane 3); orASL^(Lys3) _(UUU) (lane 4), as described under Materials and Methods.

FIG. 4. Thermal denaturation analyses of ASL^(Lys3) _(SUU) andASL^(Lys3) _(UUU). A: UV thermal denaturation profiles of the ASLs (˜2μM) were obtained as described under Materials and Methods in 10 mMphosphate buffer, pH 7.2, containing 100 mM NaCl and 0.1 mM EDTA. B:Imino proton NMR spectra of ASL^(Lys3) _(SUU) and ASL^(Lys3) _(UUU) as afunction of temperature. The spectra were collected on ˜0.1 mM ASLs in10 mM phosphate buffer, pH 6.0 containing 0.1 mM EDTA.

FIG. 5. A: Comparison of the 500-MHz DQF-COSY spectra of ASL^(Lys3)^(UUU) and ASL^(Lys3) _(SUU) in the region showing cross peaks betweenH5-H6 protons. Boxes, placed behind the cross-peaks in the ASL^(Lys3)^(UUU) spectrum and in the corresponding positions in ASL^(Lys3) _(SUU)spectrum facilitate comparison. B: Shaded boxes indicate stem pyrimidineH5-H6 cross-peaks that were not affected by the s²U-modification.Cross-peaks observed outside the boxes in the ASL^(Lys3) _(SUU) spectrumwere from the loop pyrimidines of ASL^(Lys3) _(SUU) and due to a dynamicequilibrium of the loop this ASL. A small impurity of ASL^(Lys3) _(UUU)(<10%) in the ASL^(Lys3) _(SUU) sample was responsible for weakcross-peaks inside open boxes in the ASL^(Lys3) ^(SUU) spectrum.

FIG. 6 is essentially the same as FIG. 2 above, but compared humanASL^(Lys) _(UUU) with T⁶A₃₇ with the unmodified ASL^(Lys) _(UUU) withA₃₇. Again, significantly different ribosomal binding was found.

DETAILED DESCRIPTION OF THE INVENTION

The term “microbe” as used herein includes bacteria, yeast, fungi andprotozoans. Bacteria, including both gram positive and gram negativebacteria, are preferred. Examples of suitable bacteria include, but arenot limited to, Escherichia coli, Staphylococcus aureus, andHelicobacter pylon.

The term “host” as used herein refers to human or animal cells ortissues in vitro and human or animal subjects (e.g., avian or mammaliancells, tissues and subjects such as chickens, turkeys, mouse, rat, cats,dogs, cows, pigs, horses, etc.).

The term “ribosome” as used herein refers to both intact activeribosomes and ribosome subunits that retain tRNA binding, such as 30Ssubunits.

The specific tRNA referred to herein with respect to microbe tRNA ispreferably a unique or unusual tRNA: that is, one that contains one ormore modified bases other than adenine, guanine, cytosine, or uracil inthe anticodon binding region (including both the stem and loop thereof),and/or preferably one that is the only tRNA available in that microbefor binding to a corresponding amino acid (e.g., lysine) during proteintranslation in the corresponding microbe. Preferably the modified baseor bases is/are a nucleotide(s) that is/are at a binding site asdescribed below (e.g., nucleotides 27 through 43) and participates inthe binding event. Where carried out in vivo, the tRNA for thecorresponding amino acid bound by the microbe tRNA preferably does nothave the same modified base at the binding site or correspondingnucleotide in the host organism (i.e., pathogen specific modification).Many of these exist in the human host and in agronomically importantanimal hosts as set forth above). Examples of modified bases are setforth in Table 1 below.

TABLE 1 Standard and modified nucleosides in tRNA and their standardabbreviations. U uridine C cytidine A adensoine G guanosine T thymidine?A unknown modified adenosine m1A 1-methyladenosine m2A2-methyladenosine i6A N⁶-isopentenyladenosine ms2i6A2-methylthio-N⁶-isopentenyladenosine m6A N⁶-methyladenosine t6AN⁶-threonylcarbamoyladenosine m6t6AN⁶-methyl-N⁶-threonylcarbomoyladenosine ms2t6A2-methylthio-N⁶-threonylcarbamoyladenosine Am 2′-O-methyladenosine IInosine m1I 1-methylinosine Ar(p) 2′-O-(5-phospho)ribosyladenosine io6AN⁶-(cis-hydroxyisopentenyl)adenosine ?C Unknown modified cytidine s2C2-thiocytidine Cm 2′-O-methylcytidine ac4C N⁴-acetylcytidine m5C5-methylcytidine m3C 3-methylcytidine k2C lysidine f5C 5-formylcytidinf5Cm 2′-O-methyl-5-formylcytidin ?G unknown modified guanosine Gr(p)2′-O-(5-phospho)ribosylguanosine m1G 1-methylguanosine m2GN²-methylguanosine Gm 2′-O-methylguanosine m22G N²N²-dimethylguanosinem22Gm N²,N²,2′-O-trimethylguanosine m7G 7-methylguanosine fa7d7Garchaeosine Q queuosine manQ mannosyl-queuosine galQgalactosyl-queuosine yW wybutosine o2yW peroxywybutosine ?U unknownmodified uridine mnm5U 5-methylaminomethyluridine s2U 2-thiouridine Um2′-O-methyluridine s4U 4-thiouridine ncm5U 5-carbamoylmethyluridinemcm5U 5-methoxycarbonylmethyluridine mnm5s2U5-methylaminomethyl-2-thiouridine mcm5s2U5-methoxycarbonylmethyl-2-thiouridine cmo5U uridine 5-oxyacetic acidmo5U 5-methoxyuridine cmnm5U 5-carboxymethylaminomethyluridine cmnm5s2U5-carboxymethylaminomethyl-2-thiouridine acp3U3-(3-amino-3-carboxypropyl)uridine mchm5U5-(carboxyhydroxymethyl)uridinemethyl ester cmnm5Um5-carboxymethylaminomethyl-2′-O-methyluridine ncm5Um5-carbamoylmethyl-2′-O-methyluridine D Dihydrouridine Ψ pseudouridinem1Ψ 1-methylpseudouridine Ψm 2′-O-methylpseudouridine m5U ribosylthyminem5s2U 5-methyl-2-thiouridine m5Um 5,2′-O-dimethyluridine See Sprinzl etal., Nucleic Acids Res. 26, 148 (1998).

The specific tRNA referred to herein with respect to host tRNA is alsopreferably a unique or unusual tRNA: that is, one that contains one ormore modified bases other than adenine, guanine, cytosine, or uracil inthe anticodon binding region (including both the stem and loop thereof),as set forth above, and/or preferably one that is the only tRNAavailable in that host for binding to retroviral RNA for priming ofreverse transcription of that retroviral RNA in the host.

The region of the tRNA to which binding occurs as described herein is,in general, the tRNA anticodon stem-loop structure, and most preferablythe loop structure itself. Following conventional tRNA nucleotidenumbering (see, e.g., M. Sprinzl et al., Compilation of tRNA sequencesand sequences of tRNA genes, Nucleic Acids Res. 26, 148-153 (1998)), thesite to which binding occurs is from nucleotide 27 or 32 to nucleotide39, 41 or 43. Nucleotides 32, 34, 35, 37 and 39 are preferred bindingsites, and nucleotides 34 and 37 are particularly preferred bindingsites. Binding may be to a single site or combination of sitescomprising nucleotides within this range.

As noted above, a method of screening for compounds useful forinhibiting microbial propagation is disclosed herein. The methodinvolves contacting a specific microbial tRNA such as tRNA^(lys) _(SUU),tRNA^(gln) _(SUG), or tRNA^(glu) _(SUC) to a ribosome that binds thattRNA in the presence of the test compound. The contacting step istypically carried out in vitro in an aqueous solution, with the tRNA,the ribosome, an appropriate messenger RNA, and the test compound in theaqueous solution. The contacting step may be carried out with a singletest compound or with a library of probes or test compounds in any of avariety of combinatorial chemistry systems, as discussed in greaterdetail below.

After the contacting step, the next step involves determining whetherthe compound inhibits the binding of the specific tRNA to the ribosome(e.g., the binding of tRNA^(lys) _(SUU) at position 34 thereof to theribosome).

The determining step can be carried out by any suitable means, such asthe filter binding assays disclosed below, or in any of the bindingdetection mechanisms commonly employed with combinatorial libraries ofprobes or test compounds as discussed below. Inhibition of ribosomalbinding by the test compound indicates that the test compound is usefulfor inhibiting microbial (e.g., bacterial, protozoal, fungal)propagation. Compounds identified by this technique are sometimesreferred to as “active compounds” herein. The method is particularlyuseful for identifying compounds that inhibit bacterial growth,preferably bacteria that contain a single tRNA for a particular aminoacid, such as a single lysine tRNA, particularly where the lysine tRNAcontains a 2-thiouridine at position 34. Examples of such bacteriainclude, but are not limited to, Helicobacter pylori, Escherichia coliand Staphylococcus aureus. Examples of such 2-thiouridine bases include,but are not limited to:

A method of screening for compounds useful for inhibiting retroviruspropagation in a host for the retrovirus, particularly where theretrovirus primes reverse transcription in the host by binding of aspecific host tRNA to retrovirus RNA at a pair of separate binding siteson the host tRNA, is also disclosed herein. The method comprisescontacting the specific host tRNA to the retrovirus RNA in the presenceof the test compound. The contacting step is typically carried out invitro in an aqueous solution, with the tRNA, the retroviral RNA, and thetest compound in the aqueous solution. The term “retroviral RNA” isintended to encompass both a complete retroviral genome and fragmentsthereof that contain the tRNA binding portions (such fragments willtypically be at least 10 or 12 to 50 or more nucleotides in length). Thecontacting step may again be carried out with a single test compound orwith a library of probes or test compounds in any of a variety ofcombinatorial chemistry systems, as discussed in greater detail below.

After the contacting step, the next step involves determining whetherthe compound inhibits the binding of the specific host tRNA to theretrovirus RNA in the presence of the test compound. The determiningstep can be carried out by any suitable means, such as gel shift assays,chemical and enzymatic footprinting, circular dichroism and NMRspectroscopy, equilibrium dialysis, or in any of the binding detectionmechanisms commonly employed with combinatorial libraries of probes ortest compounds as discussed below. The inhibition of binding indicatesthat the test compound is useful for inhibiting propagation of the virusin the host. Such compounds are also sometimes referred to as “activecompounds” herein. The method may be carried out with retroviruses ingeneral, particularly lentiviruses such as HIV-1. In one embodiment thespecific host tRNA is mammalian, preferably primate or specificallyhuman, such as tRNA^(lys) _(SUU), and the determining step comprisesdetermining whether the compound inhibits the binding of tRNA^(lys)_(SUU) (for example at position 34 thereof) to the retrovirus RNA.

As noted above, the present invention can be used with test compounds(or “probe molecules”), or libraries (where groups of different probemolecules are employed), of any type. In general, such probe molecules(including those that are active compounds herein) are organiccompounds, including oligomers such as antisense oligonucleotides,non-oligomers, organo-metallic compounds, and combinations thereof, aswell as bio-inorganic compounds. Non-oligomers include a wide variety oforganic molecules, such as heterocyclics, aromatics, alicyclics,aliphatics and combinations thereof, comprising steroids, antibiotics,enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids,benzodiazepenes, terpenes, prophyrins, toxins, and combinations thereof.Oligomers include peptides (that is, oligopeptides) and proteins,oligonucleotides such as DNA, RNA and their derivatives such as peptidenucleic acid (PNA), oligosaccharides, polylipids, polyester, polyamides,polyurethans, polyureas, polyethers, poly(phosphorus derivatives) suchass phosphates, phosphonates, phosphoramides, phosphonamides,phosphites, phosphinamides, etc., poly(sulfur derivatives) such assulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., wherefor the phosphorous and sulfur derivatives the indicated heteroatom forthe most part will be bonded to C, H, N, O or S, and combinationsthereof. Numerous methods of synthesizing or applying such probemolecules on solid supports (where the probe molecules may be eithercovalently or non-covalently bound to the solid support) are known, andsuch probe molecules can be made in accordance with procedures known tothose skilled in the art. See, e.g., U.S. Pat. No. 5,565,324 to Still etal., U.S. Pat. No. 5,284,514 to Ellman et al., U.S. Pat. No. 5,445,934to Fodor et al. (the disclosures of all United States patents citedherein are to be incorporated herein by reference in their entirety); J.Baldwin and I. Henderson, Recent Advances in the Generation ofSmall-Molecule Combinatorial Libraries: Encoded Split Synthesis andSolid-Phase Synthetic Methodology, Med. Res. Reviews 16, 391-405 (1996).

Such probe molecules or active compounds could be used as inhibitors bycontacting the tRNA, the RNA to which the tRNA binds (mRNA, viral RNA)or the modification enzyme responsible for the unique or unsualchemistry or structure of the tRNA (i.e., the modified base).

A method of inhibiting microbe propagation comprises inhibitingribosomal binding of a specific microbial tRNA (e.g., tRNA^(lys) _(SUU),for example at position 34 thereof) in the microbe by an amountsufficient to inhibit microbe propagation. Inhibition of ribosomalbinding may be carried out by contacting an active compound to theribosome in an amount effective to inhibit binding suffuciently toinhibit microbe propagation. The microbe is preferably a bacteria, andmost preferably a bacteria that contains a single lysine tRNA, andwherein the lysine tRNA contains a 2-thiouridine (this term includingderivatives thereof) at position 34 thereof. Examples include but arenot limited to Escherichia coli and Staphylococcus aureus. The microbemay be in vitro, in a culture media, or on a surface to be disinfected,or may be in vivo in a host (e.g., a human or animal host in need of anantimicrobial treatment). Formulations of active compounds can beprepared and administered in accordance with known techniques, asdiscussed below.

A method of inhibiting retrovirus propagation in a host for thatretrovirus, wherein the retrovirus primes reverse transcription bybinding of a specific host tRNA to retrovirus RNA at a pair of separatebinding sites on the host tRNA, comprises inhibiting the binding of thespecific host tRNA to the retrovirus RNA at one of the binding sites byan amount sufficient to inhibit propagation of the retrovirus in thehost. Note that mutation of the retrovirus RNA to bind an alternate hosttRNA for priming of reverse transcription requires a pair of mutationsat separate tRNA binding sites in the retrovirus RNA. Formulations ofactive compounds can be prepared and administered in accordance withknown techniques, as discussed below. In a preferred embodiment, thespecific host tRNA is tRNA^(lys) _(SUU), where binding may for examplebe inhibited at position 34 thereof. Preferably the retrovirus primesreverse transcription in the host specifically with the specific hosttRNA, such as tRNA^(lys) _(SUU). The method may be carried out withretroviruses in general, particularly lentiviruses such as HIV-1. Thehost may be a cell in vitro, or a human or animal subject in need ofsuch treatment.

Subjects to be treated by the methods of the present invention aretypically human subjects although the methods may be carried out withanimal subjects (dogs, cats, horses, cattle, etc.) for veterinarypurposes. The present invention provides pharmaceutical formulationscomprising the active compounds, including pharmaceutically acceptablesalts thereof, in pharmaceutically acceptable carriers for aerosol,oral, and parenteral administration as discussed in greater detailbelow. The therapeutically effective dosage of any specific compound,the use of which is in the scope of present invention, will varysomewhat from compound to compound, patient to patient, and will dependupon the condition of the patient and the route of delivery.

In accordance with the present method, an active compound or apharmaceutically acceptable salt thereof, may be administered orally orthrough inhalation as a solid, or may be administered intramuscularly orintravenously as a solution, suspension, or emulsion. Alternatively, thecompound or salt may also be administered by inhalation, intravenouslyor intramuscularly as a liposomal suspension. When administered throughinhalation the active compound or salt should be in the form of aplurality of solid particles or droplets having a particle size fromabout 0.5 to about 5 microns, preferably from about 1 to about 2microns.

The present invention also provides new pharmaceutical compositionssuitable for intravenous or intramuscular injection. The pharmaceuticalcompositions comprise an active compound or a pharmaceuticallyacceptable salt thereof, in any pharmaceutically acceptable carrier. Ifa solution is desired, water is the carrier of choice with respect towater-soluble compounds or salts. With respect to the water-insolublecompounds or salts, an organic vehicle, such as glycerol, propyleneglycol, polyethylene glycol, or mixtures thereof, may be suitable. Inthe latter instance, the organic vehicle may contain a substantialamount of water. The solution in either instance may then be sterilizedin any suitable manner, preferably by filtration through a 0.22 micronfilter. Subsequent to sterilization, the solution may be filled intoappropriate receptacles, such as depyrogenated glass vials. Of course,the filling should be done by an aseptic method. Sterilized closures maythen be placed on the vials and, if desired, the vial contents may belyophilized.

In addition to active compounds or their salts, the pharmaceuticalcompositions may contain other additives, such as pH adjustingadditives. In particular, useful pH adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the compositions may contain anti-microbial agents.Useful antimicrobial agents include methylparaben, propylparaben, andbenzyl alcohol. The microbial preservative is typically employed whenthe formulation is placed in a vial designed for multidose use. Ofcourse, as indicated, the pharmaceutical compositions of the presentinvention may be lyophilized using techniques well known in the art.

In yet another aspect of the present invention, there is provided aninjectable, stable, sterile composition comprising an active compound ora salt thereof, in a unit dosage form in a sealed container. Thecompound or salt is provided in the form of a lyophilizate which iscapable of being reconstituted with a suitable pharmaceuticallyacceptable carrier to form a liquid composition suitable for injectionthereof into man. The unit dosage form typically comprises from about 10mg to about 10 grams of the compound or salt. When the compound or saltis substantially water-insoluble, a sufficient amount of emulsifyingagent which is physiologically acceptable may be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Other pharmaceutical compositions may be prepared from the activecompounds, such as aqueous base emulsions. In such an instance, thecomposition will contain a sufficient amount of pharmaceuticallyacceptable emulsifying agent to emulsify the desired amount of theactive compound or salt thereof. Particularly useful emulsifying agentsinclude phosphatidyl cholines, and lecithin.

Further, the present invention provides liposomal formulations of theactive compounds or salts thereof. The technology for forming liposomalsuspensions is well known in the art. When the active compound or saltthereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired active compound or a saltthereof or a plurality of solid particles of the compound or salt. Thedesired formulation may be placed in a small chamber and nebulized.Nebulization may be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles should have a particle size in the range of about 0.5 to about5 microns. The solid particles can be obtained by processing thecompound, or a salt thereof, in any appropriate manner known in the art,such as by micronization. Most preferably, the size of the solidparticles or droplets will be from about 1 to about 2 microns. In thisrespect, commercial nebulizers are available to achieve this purpose.

Preferably, when the pharmaceutical formulation suitable foradministration as an aerosol is in the form of a liquid, the formulationwill comprise a water-soluble active compound or a salt thereof, in acarrier which comprises water. A surfactant may be present which lowersthe surface tension of the formulation sufficiently to result in theformation of droplets within the desired size range when subjected tonebulization.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

1. Materials and Methods

Native tRNAs, transcripts, ASLs and 30S ribosomal subunits. Native yeasttRNA^(Phe) _(GmAA) , E. coli tRNA^(Lys) _(SUU), and E. coli tRNA^(Glu)_(SUC) were purchased from Sigma Chemical Company. In vitro transcriptsof human tRNA^(Lys3) _(UUU) , E. coli tRNA^(Glu) _(UUC) and E. colitRNA^(Gln) _(UUC) were gifts from Drs. Musier-Forsyth and Rodgers andSöll, respectively. ASLs, with and without s²U, were synthesized usingan adaptation of standard solid phase chemistry for RNA synthesis andpurified by HPLC (Agris et al., Biochimie 77: 125-134 (1995); Kumar andDavis, Nucleic Acids Res. 25: 1272-1280(1997)). Sequence andmodification of the ASLs were confirmed by MALDI mass spectroscopy(Faulstich et al., Anal. Chem. 69: 4349-4353 (1997)), nucleosidecomposition analysis (Gehrke and Kuo, Chromatography and modification ofnucleoside Vol. 45A, ppA3-A71 (Amsterdam: Elsevier 1990) andorganomercurial-gel electrophoresis (Igloi, Biochemistry 27: 3842-3849(1998)). Both mass spectroscopy and HPLC analyses confirmed the purityof ASL^(Lys3) _(SUU) to be greater than 90%. Thermal denaturation andrenaturing of tRNA transcripts and ASLs did not alter their abilities tobind the 30S subunits. Small ribosomal subunits (30S) were prepared andactivated according to Ericson et al. (J. Mol. Biol. 250: 407-419(1995)).

Binding of tRNAs and ASLs to 30S ribosomal subunits. In order todetermine the K_(d) of tRNA and ASL binding to the ribosome, 30Ssubunits (10 pmol) and poly(U), poly(A), poly(AG) or poly(AC) (10 μg)were incubated for 20 minutes at 37° C. with increasing amounts of³²P-labeled tRNA or ASL (up to 50 pmol) in 40 ml of CMN buffer (80 mMpotassium-cacodylate, pH 7.2, 20 MM MgCl₂, 100 mM NH₄Cl, and 3 mMβ-mercaptoethanol). The reaction was incubated for another 20 minutes onice, passed through nitrocellulose filters (0.45 μm) and thefilter-bound RNA washed twice with ice-cold buffer (100 μl ). Thefilters were then air-dried and counted. K_(d)s and their standarddeviations were determined using non-linear regression analysis on fourreplicates of each experiment.

Chemical modification and primer extension. Chemical probing and primerextension was accomplished as described by Moazed and Noller (J. Mol.Biol. 211: 135-145 (1990)) except that the modifications with bothdimethyl sulfate and kethoxal were conducted at 20° C. for 30 minutes in40 ml of CMN buffer. Ribosomal 30S subunits (10 pmol) programmed withpoly(U), poly(A), or poly(AG) (10 mg) were incubated with 50 pmolesyeast tRNA^(Phe) _(GmAA) or ASL^(Phe) _(GAA or) 150 pmoles of E. colitRNA^(Lys) _(SUU), human ASL^(Lys3) _(SUU), or human ASL^(Lys3) _(UUU).

UV and NMR Spectroscopy. Thermal denaturations of tRNA^(Lys) ASLs weremonitored with UV and NMR spectroscopies. The UV samples contained ˜2 M(0.3 A₂₆₀ units) RNA in 10 mM sodium phosphate buffer, pH 7.2, 100 mMNaCl and 0.1 mM EDTA. NMR samples contained ˜0.1 mM (11.5 A₂₆₀ units)RNA in 10 mM phosphate buffer, pH 6.0, and 0.1 mM EDTA. Double quantumfiltered COSY (DQF-COSY) experiments were conducted at 10° C. with aBruker DRX 500 MHz spectrometer using standard procedures (Rance et al.,Biochem. Biophys. Res. Commun 117: 479-485 (1983)). The exchangeable andnon-exchangeable proton resonances were assigned using standardprocedures except for the imino protons of U₃₄, U₃₅ and U₃₆ which wereassigned via site-specific incorporation of ¹⁵N3-uridines. Resonances ofASL_(Lys) _(SUU) were assigned by comparison to the spectra of ASL^(Lys)_(SUU).

2. Results

Ribosomal Binding of Native tRNAs, Unmodified tRNAs, and ASLs. Theunmodified anticodon stem and loop of E. coli tRNA^(Lys) _(UUU)(ASL^(Lys) _(UUU)) and that of tRNA^(Gln) _(UUG) (ASL^(Gln) _(UUG)) didnot bind the appropriately programmed ribosomes, presumably because oflack of modified nucleosides (von Ahsen et al., RNA 3: 49-56 (1997)).However, rather than requiring one or more modified nucleosides, perhapsthese particular ASLs failed to bind because their smaller sequenceslacked a required structural element. Therefore, we compared theribosome binding of the unmodified transcripts of the tRNAs to that offully modified, native tRNAs. In vitro transcribed, full-length E. colitRNA^(Gln) _(UUG) and tRNA^(Glu) _(UUC) and human tRNA^(Lys3) _(UUU) allfailed to bind the appropriately programmed 30S ribosomal subunits(FIGS. 2A and B). Under the same conditions, native E. coli tRNA^(Lys)_(SUU) effectively bound poly(A)-programmed ribosomes (FIG. 2A), E. colitRNA^(Glu) _(SUC) bound randomly polymerized poly(AG)-programmedribosomes (FIG. 2B), and yeast tRNA^(Phe) _(GmAA) boundpoly(U)-programmed ribosomes (FIG. 2C). Thus, the lack of ribosomolbinding by unmodified E. coli lysine and glutamine ASLs (von Ahsen etal., supra) was not due to the size of the RNA used in theseexperiments. A similar negative result was observed for the transcriptof tRNA^(Lys) _(UUU) when ribosomes were programmed with randomizedpoly(AG) containing both the AAG and AAA codons (data not shown). Thus,the inability of unmodified ASLs and tRNA transcripts of lysine,glutamine, and glutamic acid to bind the ribosome was neither due to thesequence length nor a codon preference. Because of these results and thefact that fully modified tRNA^(Lys) _(SUU), tRNA^(Gln) _(SUG) andtRNA^(Glu) _(SUC) have in common s²U derivatives at the wobble position34, we postulated that s²U₃₄-containing-tRNAs are dependent onnucleoside modifications for ribosome binding.

In order to test this hypothesis, heptadecamer ASLs corresponding to thehuman tRNA^(Lys3) _(SUU) sequence were produced with and without s²U₃₄by automated oligonucleotide synthesis and then assayed for ribosomebinding. Human and E. coli tRNA^(Lys) _(SUU) ASLs have identicalsequences except for the three base pairs at the top of the stem (Agriset al., RNA 3: 420-428 (1997)). Surprisingly, we found that theASL^(Lys3) _(SUU) (FIG. 1A), singularly modified with just s²U atposition 34, was able to bind poly(A)- and poly(AG)-programmed ribosomesalmost as effectively as native E. coli tRNA^(Lys) _(SUU) (FIG. 2A). Infact, the K_(d) for the interaction of ASL^(Lys3) _(SUU) withpoly(A)-programmed 30S ribosomal subunits (176±62 nM) was comparable tothat of native E. coli tRNA^(Lys) _(SUU) (70±7 nM). As expected, theunmodified human ASL^(Lys3) _(UUU) (FIG. 1A) did not bind AAA- orAAG-programmed ribosomes (FIG. 2A). In contrast, unmodified yeastASL^(Phe) _(GAA) bound poly(U)-programmed 30S ribosomal subunits aseffectively (K_(d)=136±49 nM) as native tRNA^(Phe) _(GmAA) (K_(d)=103±19nM) (FIG. 2C).

16S P-site footprints by tRNAs and ASLs. To determine if the ASL^(Lys3)_(SUU) bound the ribosome at the same site as fully modified tRNA^(Lys)_(SUU), we conducted a chemical footprinting analysis of the ASL andtRNA on 16S rRNA. Chemical protections were analyzed for five of thecommonly recognized 16S P-site nucleotides (A532, G926, A794, C795, andG1338) (Moazed and Noller, supra). The ASL^(Lys3) _(SUU) produced thesame footprint on 16S rRNA as did the native E. coli tRNA^(Lys) _(SUU)(FIGS. 3A and B). Both ASL and tRNA protected all five P-sitenucleosides. Of the five 16S rRNA nucleosides that were probed, A532,G926 and G1338 were intrinsically more reactive to chemical probes thanA794 and C795. Hence, they were more easily recognized as beingprotected in the presence of ASL or tRNA (FIG. 3B). The reduced chemicalreactivities and the weaker protections of A794 and C795 have also beendocumented by others (Moazed and Noller, supra). As expected, theunmodified ASL^(Lys3) _(UUU) provided no substantial protection ofP-site nucleotides from either kethoxal (FIG. 3A) or dimethyl sulfate(FIG. 3B) chemical probes. In contrast, the unmodified yeast ASL^(Phe)_(GAA) produced the same footprint as did native yeast tRNA^(Phe)_(GmAA), the ASL^(Lys3) _(SUU) and native E. coli tRNA^(Lys) _(SUU).Thus, as shown by both filter binding and chemical probing, for sometRNAs, such as tRNA^(Lys) _(SUU), but not all, the nucleosidemodifications are critical for ribosomal P-site binding.

UV and NMR Spectroscopies. The dramatic restoration of ribosomal bindingactivity by a single, simple base modification underscores theimportance of the 2-thio group in tRNA^(Lys) _(SUU) and prompted us toexamine possible structural differences between the unmodifiedASL^(Lys3) _(UUU) and the ASL^(Lys3) _(SUU). Thermal denaturations,monitored by both UV spectroscopy and the imino proton chemical shiftsof NMR spectra (FIGS. 4A and B), failed to detect any significantdifferences in the stability of the ASLs. The ASLs had similardenaturation profiles and identical melting points (T_(m)=48±1° C.).These results were not surprising considering that the techniquesemployed principally monitored the stabilities of ASL stems and not theloops. However, structural and dynamic differences between the two ASLloops became apparent in analysis of NMR spectra of non-exchangeableprotons (FIG. 5). Examination of the DQF-COSY spectra of ASL^(Lys3)_(UUU) and ASL^(Lys3) _(SUU) revealed that H5-H6 cross-peaks arisingfrom pyrimidines in the first four base pairs of the stem (U₂₇, C₂₈, C₄₀and U₄₁) were not affected by the presence of s²U₃₄ in the loop (FIGS.5A and B). However, the remaining six pyrimidines of the modifiedASL^(Lys3) _(SUU), including five in the loop (C₃₂, U₃₃, S²U₃₄, U₃₅ andU₃₆), produced more than six signals. These results suggest that theloop region of ASL^(Lys3) _(SUU), unlike that of the unmodifiedASL^(Lys3) _(UUU), was engaged in a slow conformational equilibriuminvolving two or more species. We have yet to fully describe thatequilibrium. However, there is no doubt that the presence of s²U haschanged the loop.

The results presented here are the first demonstration of a single atommodification (O→S) in tRNA that is critical for ribosomal binding.Thiolated human ASL^(Lys3) _(SUU) binds the ribosome, yet unmodified E.coli ASL^(Lys) _(UUU), the unmodified human tRNA^(Lys3) _(UUU)transcript and the corresponding ASL do not. In addition, the unmodifiedtranscripts of tRNA^(Glu) _(UUC) and tRNA^(Gln) _(UUG), as well as theunmodified ASL^(Glu) _(UUG) (von Ahsen, supra), do not bind theribosome. These results demonstrate the importance of the s²U₃₄nucleoside modification in the in vitro P-site binding of manyS²U₃₄-containing tRNAs, regardless of genus. In addition, asuE (trm U)mutants of E. coli [personal communication, Dieter Söll] and sin 3/sin 4mutants of Schizosaccharomyces pombe (Grossenbacher et al., J. biol.Chem. 261: 16351-16355 (1986); Heyer et al., J. Biol. Chem. 259:2856-2862 (1984)) deficient in the synthesis of s²U grow very poorly,suggesting ribosomal binding of s²U-containing tRNAs is modificationdependent in vivo, as well as in vitro.

To provide further evidence for nucleoside modifications being targetsfor antibacterial drugs, ribosome binding of ASL^(Lys) _(UUU) with t⁶A₃₇was compared to the unmodified ASL^(Lys) _(UUU) with A₃₇. (t⁶A isN6-threonylcarbamoyladenosine). Studies were carried out in essentiallythe same manner as described in connection with FIG. 2 above and thesedata are presented in FIG. 6. Again, the modified nucleotide, this timeat position 37, was critical for ribosome binding. These data indicatethat modified nucleoside throughout the stem-loop structure may betargeted for drug binding, interaction and screening as described above.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of screening for compounds useful for inhibiting retrovirus propagation in a host for said retrovirus, wherein said retrovirus primes reverse transcription in said host by binding of a specific host tRNA to retrovirus RNA at a pair of separate binding sites on said host tRNA, said method comprising: contacting in vitro said specific host tRNA to said retrovirus RNA in the presence of said test compound; and then; determining whether said test compound inhibits the binding of said specific host tRNA to said retrovirus RNA; the inhibition of binding indicating said test compound is useful for inhibiting propagation of said virus in said host; whereby mutation of said retrovirus RNA to bind an alternate host tRNA for priming of reverse transcription requires a pair of mutations at a pair of separate tRNA binding sites in said retrovirus RNA.
 2. A method according to claim 1, wherein said determining step comprises determining whether said compound inhibits the binding of said tRNA to said retrovirus RNA at position 27-43 of said tRNA.
 3. A method according to claim 1, wherein said specific host tRNA is tRNA^(lys) _(suu).
 4. A method according to claim 3, wherein said determining step comprises determining whether said compound inhibits the binding of tRNA^(lys) _(suu) at position 34 thereof to said retrovirus RNA.
 5. A method of screening for compounds useful for inhibiting retrovirus propagation in a host for said retrovirus, wherein said retrovirus primes reverse transcription in said host by binding of a specific host tRNA to retrovirus RNA at a pair of separate binding sites on said host tRNA, wherein said specific host tRNA is tRNA^(lys) _(suu); said method comprising: contacting in vitro said specific host tRNA to said retrovirus RNA in the presence of said test compound; and then; determining whether said compound inhibits the binding of said specific host tRNA to said retrovirus RNA at position 27-43 of said tRNA in the presence of said test compound; the inhibition of binding indicating said test compound is useful for inhibiting propagation of said virus in said host; whereby mutation of said retrovirus RNA to bind an alternate host tRNA for priming of reverse transcription requires a pair of mutations at a pair of separate tRNA binding sites in said retrovirus RNA. 